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Cellometer Image Cytometry for Cell
Cycle Analysis
Importance of Cell Cycle Research
• Oncology:
• Since cancer cells often undergo abnormal cell division and proliferation, it is important to understand the cell cycle of those cells.
• Pharmacology:
• Studies involving characterization of pharmacological reagents and their effect on cell cycle regulation
• Development of drugs to arrest normal cell cycling during chemotherapy treatment to reduce side effects
• Cell/Molecular Biology:
• To understand molecular and biological mechanisms, it is essential to examine their role in cell cycle.
What is Cell Cycle?
1. G1 (G=Gap) phase. Cells are preparing for DNA replica3on. At this stage the cells contain about the same amount of DNA. (diploid (2n))
2. S (S=Synthesis) phase. Replica3on of nuclear DNA occurs at this stage. During S phase there is a broad distribu3on of DNA content between G1 and G2 phase. The DNA content is doubled by the end of this phase. (2n to 4n)
3. G2 (G=Gap) phase. Replica3on of the DNA is complete, and the cell is growing and preparing for division. At this stage, the cells contain twice the number of chromosomes (4n) that are found in G1.
4. M (M=Mitosis) phase. Cell division.
Cell cycle is defined as “the period between successive divisions of a cell.” Standard cell cycle of eukaryotic cells is divided into 4 phases.
Cell Cycle Detection Methods
There are multiple methods and protocols for detecting DNA content during cell cycle: Radioactivity Measurement
3H-thymidine – Radioactive labeling of thymidine. Upon DNA replication, the radiolabled thymidine is incorporated into new DNA.
Fluorescence-Based Detection
• Live cell measurement Hoechst 33342 – Is cell permeable and therefore do not require cell fixation. Binds to
the minor groove of double-stranded DNA.
• Fixed cell measurement Bromodeoxyuridine (BrdU) – A thymidine analog, is incorporated into the genome
during the S-phase of the cell cycle. Detected using anti-BrdU antibodies.
DAPI – Binds to the AT rich regions of the DNA. Poor live cell permeability and therefore often used post-fixing.
Propidium Iodide (PI) – A membrane exclusion dye and an intercalating agent that stains the cellular genome upon cell fixation.
Fluorescence-based Cell Cycle Analysis using PI
Labeling DNA with propidium iodide (PI) allows for fluorescence-‐based analysis of cell cycle.
• This assay generates a cell popula3on histogram (on leL) in respect to PI fluorescence intensity.
• Amount of PI fluorescence intensity is correlated to the amount of DNA within each cell.
• Since the amount of DNA doubles (2n à 4n) between G1 and G2 phases, the amount of fluorescence intensity of the cell popula3on also doubles.
2n 4n
Cell Cycle Analysis
Control Media
24 hour Incubation
Nocodazole
Sample preparation
In this cell cycle experiment, cells were treated with Nocodazole, a compound designed to arrest the cell cycle at the G2M Phase.
Sample Preparation for PI Cell Cycle Analysis
Collect cells (control and treated)
Fix for 15 min
Stain with PI Cell Cycle Reagent (40 min)
Re-suspend in final volume of PBS
Analyze using Vision CBA
Cellometer Image Cytometry Procedure ��� Analyze Cells in Just a Few Steps
4. Click count to image and view cell count, concentration, and diameter
1. Pipette 20uL of cells into counting chamber
2. Insert chamber
6. With Vision CBA, click export to view data plots
3. Select Assay & enter Sample ID
5. View bright field and fluorescent cell images
PI Cell Cycle: Jurkat Cells
Bright Field Image
Propidium Iodide Image
Sample Information Cell type: T lymphocyte
Disease: acute T cell luekemia Type: non adherent
Untreated control
Image Cell Pop % Gated Sub G1 0.2% G0/G1 62.1% S 17.1% G2/M 15.4%
Nocodazole-Induced Cell Cycle Arrest
G0/G1
G2/M
S
Sub G1
Nocodazole [0.004 -0.1µg/ml] Dose Response Cells arrested at G2/M Phase
Control
Nocodazole (μg/ml) 0.004 0.02 0.1 6.71% 12.15% 9.18% 53.69% 10.27% 7.93% 17.26% 17.81% 9.39% 12.35% 41.40% 47.78%
Phase Control
Sub G1 6.96%
G0/G1 53.12%
S 15.00%
G2/M 14.35%
Cellometer and Flow Cytometry Correlation
% of Cells Arrested at G2/M Phase
Excellent correlation between Cellometer Vision CBA and flow results
Cellometer
Flow
Phase 0.004 μg/ml 0.02 μg/ml 0.1 μg/ml
G2/M 12.35% 41.40% 47.78%
G2/M 15.25% 39.10% 43.10%
PI Cell Cycle: PC-3 Cells
Bright Field Image
Propidium Iodide Image
Sample Information Organ: prostate
Cell Type: epithelial Disease: adenocarcinoma
Type: adherent
Untreated control
PI Cell Cycle: HeLa Cells
Sample Information Organ: cervix
Cell Type: epithelial Disease: adenocarcinoma
Type: adherent
Untreated control Image
Bright Field Image
Propidium Iodide Image
PI Cell Cycle: PANC-1 Cells
Sample Information Organ: pancreas
Cell Type: epithelial Diseases: epithelioid carcinoma
Type:adherent
Untreated control Image
Bright Field Image
Propidium Iodide Image
PI Cell Cycle: HT-29
Sample Information Organ: colon
Cell Type: epithelial Diseases: colorectal adenocarcinoma
Type: adherent
Untreated control Image
Bright Field Image
Propidium Iodide Image
DAPI as an Alternative Staining Method for Cell Cycle
Bright Field Image
UV Image
Sample Information PC3 cells: Untreated control
Organ: prostate Cell Type: epithelial
Disease: adenocarcinoma
Type: adherent
Summary
• The Cellometer can be utilized to effectively measure and analyze the cell cycle of mammalian cells.
• There is an excellent correlation between the Cellometer results and flow cytometry results.
• Cellometer may be used to examine a wide variety of mammalian cells.